PPARlox & PPARwt PCR Protocol
Kathy Shelton
The following conditions are used to genotype PPAR mouse genomic DNA screening
for a floxed (lox) allele and/or a wild type (wt) allele. The same set of PCR primers will
screen for both “G” (lox) & “W” (wild type) in a single reaction.
PCR reagents:
1. oligo primers at 20 µM
PPAR lox & wt (amplify a 572 bp “loxed” fragment and a 464 bp
“wt” fragment)
PPAR4 5’-GCT CCT GAG TGC TAA TAT TAA AG-3’ (top)
PPAR5 5’-CCA TGG ACT AAT GCT GTA ATA TTA-3’ (bottom)
Internal control primers are omitted in these PCR reactions since the animals
being screened should give a band for G, W, or G & W.
2. Perkin Elmer PCR buffer with MgCl2
3. dNTP premix (I make my own dNTP premix using 100 mM NEB dNTP’s.
The premix contains 250 µl of each dNTP - A, C, G, &T - and 19 ml sterile
water and is stable at -20°C. I freeze this in 1 ml aliquots and thaw and
refreeze as needed.)
4. Perkin Elmer Amplitaq Gold
5. genomic DNA samples diluted to 50 ng/µl with sterile water
PCR reaction mixture:
15.8 µl sterile water
2.5 µl 10X PCR buffer
4 µl dNTP premix
0.75 µl primer PPAR4
0.75 µl primer PPAR5
1 µl dil. DNA template
0.2 µl Amplitaq Gold
25 µl total volume
Cycling conditions:
1 cycle - 94°C x 6 min.
40 cycles - 94°C x 1 min., 60°C x 30 sec., 72°C x 30 sec.
1 cycle - 72°C x 7 min.
hold at 4°C.
Analysis of PCR products:
Load 10 µl aliquots of reactions + 2 µl gel loading buffer in a 1% mini-agarose gel (using
a 15-well comb). Run gel approximately half-way down.
Sample gel illustrating amplicons for PPARlox & PPARwt amplicons.
Here one can see the importance of running these samples side-by-side to aide in
distinguishing “loxed” & “wt” genotypes.
572 bp “G” band
464 bp “W” band